Localized activation of m-calpain in human umbilical vein endothelial cells upon hypoxia

Bleb formation is an early event of cellular damage observed in a variety of cell types upon hypoxia. Although we previously found the appearance of the localized cytoplasmic ionized Ca(2+) concentration ([Ca(2+)](i)) rise before bleb formation at the same loci of human umbilical vein endothelial cell (HUVEC) upon hypoxia, the mode of [Ca(2+)](i)-rise-induced cytoskeletal alteration remains ill-defined. The aim of this study is to clarify the mechanisms causing bleb formation after localized [Ca(2+)](i) rise. We studied the activation of m-calpain associated with the alteration of cytoskeleton-related proteins, F-actin, mu-actin, or ezrin by employing specific antibodies in conjunction with a confocal laser scanning microscopy (CLSM). Specific antibodies against 80-kDa-preactivated and 78-kDa-activated m-calpain clearly demonstrated redistribution of 80-kDa m-calpain followed by autoproteolytic activation of m-calpain to the 78-kDa form at the same loci of [Ca(2+)](i) rise in hypoxia-treated HUVECs, which was associated with the decrease of ezrin and the localized appearance of beta-actin at the same loci. In conclusion, hypoxia-induced localized [Ca(2+)](i) rise causes bleb formation at the same loci through m-calpain-catalyzed destruction of cross-linking between plasma membrane and actin filaments.